The major limitation to the use of gene therapy techniques in the hematopoietic system of humans is the inability to efficiently transfer genes into hematopoietic stem cells (HSC) capable of providing long- term multi-lineage engraftment. During the previous period of this SCOR grant, we improved the efficiency of gene transfer to HSC in the baboon animal model from <1% to up to 20% by using a retroviral vector with a gibbon ape leukemia virus (GALV) coat, by alteration of the conditions for transduction to include a specific cocktail of cytokines, and by using culture dishes coated with the fibronectin fragment CH-296. For the current application, we propose to test cannot transduce non-dividing cells, and it seems likely that improvements could be obtained by using lentivirus vectors capable of transducing non-dividing cells, and it seems likely that improvements could be obtained by using lentivirus vectors capable of transducing non-dividing cells, including non-dividing or slowly dividing HSC. Similarly, vectors with a VSV-G coat may be capable of higher transduction rates based on the apparent use of phosphatidylserine, an abundant membrane component, as a receptor for HSV-G-mediated vector entry. To date, however, efficient transduction of HSC of large animals has not been demonstrated by using VSV-G pseudotype lentivirus vectors. Here we propose to test whether HIV- based lentivirus vectors promote improved transduction of HSC of large animals has not been demonstrated by using HSV-G pseudotype lentivirus vectors. Here we propose to test whether HIV-based lentivirus vectors promote improved transduction of HSC in baboons, and specifically, whether a GALV pseudotype of the HIV vector provides improved transduction compared with a HSV-G pseudotype of the HIV vector. We will further investigate VSV G-mediated vector entry and attempt to identify receptors in addition to phosphatidyl serine that might be required for entry, in attempt to understand possible limitations to HSV-G mediated transduction. Finally, we have found a number of non- mammalian cell lines that show various blocks to transduction by lentivirus vectors, and we propose to study these cells to better understand the factors required for efficient lentivirus vector transduction Our ultimate test for methods that might increase transduction of stem cells will be engraftment of vector-containing cells in the baboon, studies performed in collaboration with Dr. Andrews in Project 6. In summary, for many clinical applications it is critically important to increase the rate of stem cell transduction to as close to 100% as possible, and we propose to determine the optimal vector and transduction conditions to achieve this goal.